Regulation of Abiotic Stress Responses in Plants by the Ubiquitin Pathway

该项目旨在了解植物如何应对压力环境以生存。虽然我们知道植物在暴露于寒冷、干旱和高盐度的条件下会合成一种新的化学物质，但我们不知道植物是如何“打扫房间”的，也就是说，压力信号是如何被移除的，这样一旦压力消失，植物就能开始正常生长。遗传学和生物化学方法都将被用来确定这些变化是如何发生的。这项研究将通过研究项目为加州大学戴维斯分校的多名本科生提供培训，并支持一名研究生和一名博士后学者。这些人将学习科学方法，以及如何向同事和更大的社区展示科学数据。他们将学习分析技巧，提高口语和写作能力。研究生将有机会与拥有大量西班牙裔人口的邻近社区的K-12学校进行互动。研究人员将通过加州大学戴维斯分校种子研究所与加州种子/农业生物技术产业的代表进行互动。研究人员已经与萨克拉门托当地的一位高中老师建立了联系，并将继续这项合作。高中生将学习实验设计和数据分析，并参与科学发现。植物通过上调帮助它们应对环境压力的基因表达来应对环境压力。应激反应所需的一个转录因子家族被称为ABRE结合蛋白（ABFs），并在蛋白质水平上受到调节。在压力下，它们被激活以促进转录，同时它们的降解被停止，蛋白质水平增加。应激如何引起abf的这两种变化及其与生物学功能的关系尚不清楚。在正常生长条件下，ABF氨基酸负责调节构成ABF蛋白水解和应激诱导的蛋白质稳定，将通过体外和体内降解试验来确定。编码ABF蛋白的DNA序列将被改变，因此它们翻译ABF蛋白时缺失和/或氨基酸替换。ABF片段还将与标记酶的编码区融合，并通过快速定量分析确定融合的稳定性。改变的蛋白质将从细菌中表达和纯化，并在植物裂解物中测量稳定性。将序列导入植物，产生多个表达这些蛋白的稳定系，并测定其在体内的稳定性。磷酸化状态的作用将通过测量用不能修饰的丙氨酸或模拟修饰的天冬氨酸取代的预测或可能的磷酸化位点的蛋白质的稳定性来评估。将对表达降解率不同的ABF蛋白的转基因植物在正常和胁迫条件下的生长表型进行评估，以确定降解调节是否能在不影响正常生长的情况下提供更强的抗逆性。

This project aims to understand how plants respond to a stressful environment in order to survive. While it is known that plants synthesize a novel chemical when exposed to conditions such as cold, drought and high salinity, what is not understood is how the plant "cleans house", that is, how the stress signal is removed so that the plant can begin normal growth once the stress is gone. Both genetic and biochemical approaches will be taken to determine exactly how these changes happen. This research will provide training of multiple UC-Davis undergraduates through research projects and support a graduate student and a post-doctoral scholar. These individuals will learn scientific methodology and how to present scientific data to colleagues and the greater community. They will learn analytical skills and improve their speaking and writing abilities. The graduate student will have the opportunity to interact with K-12 schools in neighboring communities with large Hispanic populations. The researcher will interact with representatives of the California seed/agricultural biotechnology industries through the Seed Institute at UC-Davis. The researcher has established a connection with a local Sacramento high school teacher and will continue this collaboration. The high school students will learn experimental design and data analysis, and participate in scientific discovery. Plants respond to environmentally stressful conditions by upregulating expression of genes that help them cope. One family of transcription factors required for stress responses are called ABRE BINDING PROTEINS (ABFs), and are regulated at the protein level. Under stress they are activated to promote transcription and at the same time their degradation is stopped and protein levels increase. How stress causes these two changes in ABFs and their relationship to biological function are not understood. ABF amino acids responsible for regulating constitutive ABF proteolysis under normal growth conditions and for stress-induced protein stabilization will be determined through in vitro and in vivo degradation assays. DNA sequences encoding ABF proteins will be changed so they translate ABF proteins with deletions and/or amino acid substitutions. ABF segments will also be fused with coding regions of marker enzymes and stability of the fusion determined in rapid and quantitative assays. Altered proteins will be expressed and purified from bacteria and stability measured in plant lysates. Sequences will be introduced into plants, multiple stable lines expressing these proteins produced and the in vivo protein stability measured. The role of phosphorylation status will be assessed by measuring the stability of proteins with substituted predicted or possible phosphorylation sites with either alanine, which cannot be modified, or with aspartate, which mimics modification. Transgenic plants expressing ABF proteins with differences in degradation rates will be assessed for their growth phenotypes under normal and stress conditions to determine if modulation of degradation provides greater stress resistance without compromising normal growth.